Yoshiaki Nishiya

Reactivation of Insertionally Inactivated Shiga Toxin 2 Genes of Escherichia coli O157:H7 Caused by Nonreplicative Transposition of the Insertion Sequence

ABSTRACT IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes of Escherichia coliO157:H7. We analyzed the transpositional mechanism of IS1203v in order to investigate whether the Shiga toxin 2 genes inactivated by IS1203v could revert to the wild type. When the transposase activity of IS1203v was enhanced by artificial frameshifting, IS1203v was obviously excised from the Shiga toxin 2 gene in a circular form. The IS1203v circle consisted of the entire IS1203v, but an extra 3-bp sequence (ATC) intervened between the 5′ and 3′ ends of IS1203v. The extra 3-bp sequence was identical to a direct repeat which was probably generated upon insertion. Moreover, we detected the Shiga toxin 2 gene with a precise excision of IS1203v. In the wild-type situation, the transposition products of IS1203v could be observed by PCR amplification. These results show that IS1203v can transpose in a nonreplicative manner and that the Shiga toxin gene inactivated by this insertion sequence can revert to the wild type.

Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity

Bacillus sp. TB-90 urate oxidase (BTUO) is one of the most thermostable homotetrameric enzymes. We previously reported [Hibi, T., et al. (2014) Biochemistry 53, 3879-3888] that specific binding of a sulfate anion induced thermostabilization of the enzyme, because the bound sulfate formed a salt bridge with two Arg298 residues, which stabilized the packing between two β-barrel dimers. To extensively characterize the sulfate-binding site, Arg298 was substituted with cysteine by site-directed mutagenesis. This substitution markedly increased the protein melting temperature by ∼ 20 °C compared with that of the wild-type enzyme, which was canceled by reduction with dithiothreitol. Calorimetric analysis of the thermal denaturation suggested that the hyperstabilization resulted from suppression of the dissociation of the tetramer into the two homodimers. The crystal structure of R298C at 2.05 Å resolution revealed distinct disulfide bond formation between the symmetrically related subunits via Cys298, although the Cβ distance between Arg298 residues of the wild-type enzyme (5.4 Å apart) was too large to predict stable formation of an engineered disulfide cross-link. Disulfide bonding was associated with local disordering of interface loop II (residues 277-300), which suggested that the structural plasticity of the loop allowed hyperstabilization by disulfide formation. Another conformational change in the C-terminal region led to intersubunit hydrogen bonding between Arg7 and Asp312, which probably promoted mutant thermostability. Knowledge of the disulfide linkage of flexible loops at the subunit interface will help in the development of new strategies for enhancing the thermostabilization of multimeric proteins.

Simple and reliable urea assay based on a signal accumulation type of ion-sensitive field-effect transistor

A simple urea assay was developed using a signal accumulation type of ion-sensitive field-effect transistor (SA-ISFET). Decreases in proton concentration resulting from urease-catalyzed hydrolysis of urea are detected by SA-ISFET as a change in potential. The method exhibits high sensitivity, linearity, and reproducibility when potential signals are accumulated 10-fold.

Over-expression, characterization, and modification of highly active alkaline phosphatase from a Shewanella genus bacterium

We isolated a Shewanella sp. T3-3 bacterium that yielded highly active alkaline phosphatase (APase). We then cloned the APase gene from Shewanella sp. T3-3 (T3-3AP), and expressed and purified the enzyme from Escherichia coli. Recombinant T3-3AP showed high comparative reactivity on colorimetric (pNPP) and luminescent substrates (PPD and ASP-5). Subsequently, we improved the residual activity after maleimide activation by introducing amino acid substitutions of two Lys residues that were located near the active site. The double mutant enzyme (K161S + K184S) showed much higher residual specific activity after maleimide activation than the wild type enzyme, and had approximately twofold increased sensitivity on sandwich enzyme linked immunosorbent assays (ELISA) compared with calf intestinal APase (CIAP), which is routinely used as a labeling enzyme for ELISA. Finding of new APase that is superior to a commercial enzyme used routinely, CIAP, and enzyme improvement to increase sensitivity on sandwich ELISA.

Characterization of a thermostable glucose dehydrogenase with strict substrate specificity from a hyperthermophilic archaeon Thermoproteus sp. GDH-1.

A hyperthermophilic archaeon was isolated from a terrestrial hot spring on Kodakara Island, Japan and designated as Thermoproteus sp. glucose dehydrogenase (GDH-1). Cell extracts from cells grown in medium supplemented with glucose exhibited NAD(P)-dependent glucose dehydrogenase activity. The enzyme (TgGDH) was purified and found to display a strict preference for d-glucose. The gene was cloned and expressed in Escherichia coli, resulting in the production of a soluble and active protein. Recombinant TgGDH displayed extremely high thermostability and an optimal temperature higher than 85 °C, in addition to its strict specificity for d-glucose. Despite its thermophilic nature, TgGDH still exhibited activity at 25 °C. We confirmed that the enzyme could be applied for glucose measurements at ambient temperatures, suggesting a potential of the enzyme for use in measurements in blood samples. Graphical abstract The glucose dehydrogenase which we obtained (TgGDH) has both high thermal stability and high substrate specificity.